Ibrahim Elhaj

Ibrahim Elhaj

31 Part 1

Mamoun Ahram

Nabil G. Sweis

Faculty of medicine – JU2018

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❖ Quick Introduction:

➢ Proteomics: a field that allows us to study proteins in cells.

➢ The objective of this field is to identify and analyze a lot of different proteins

present in living organisms, not just a single protein.

➢ One of the techniques used in this field is “2-dimensional gel electrophoresis”.

➢ However, 2D gel electrophoresis has a prominent limitation:

✓ We have -on average- 6000 genes expressed in cells, many of the gene

products (proteins) are modified (post-translational modifications such as

glycosylation, phosphorylation, cleavage…etc). According to scientists’

estimations, 100,000 different proteins emerge from these 6000 genes

(notice that it is NOT a 1-to-1 ratio due to splicing and other modifications

that diversify gene products).

✓ From these 100,000 proteins, 2D gel electrophoresis enables us to identify

around 1000 proteins which corresponds to merely 1% of the total number

of different proteins we actually have.

✓ This limitation called for the emergence of other proteomic techniques that

would assist us in identifying a larger variety of proteins, which we will

discuss next.

❖ Mass Spectrometry:

✓ How does it work?

➢ Proteins are digested/cleaved into peptides (usually by using

trypsin), separated by chromatography and injected into the mass

spectrometer (a large instrument) where they get ionized and

travel according to size and charge and reach a detector.

➢ The speed of travel is measured, and then with the help of

bioinformatics, we translate this piece of information into a

protein sequence or peptide identity.

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Please return to the animation which you can find in the “Lecture 31" video (starting at 3:10)

➢ Mass spectrometry was even further developed to what is known as “tandem

mass spectrometry”. (remember: tandem = one after the other).

➢ In tandem mass spectrometry, you have two mass spectrometers placed in

succession (one after the other). After passing the first mass spectrometer, one of

the peptides that reaches the detector in the first mass spectrometer is further

degraded into smaller pieces and ionized again in the second mass spectrometer,

and these new smaller pieces travel within the second mass spectrometer where

they reach a new detector yielding new information. This new information

uncovers even more detail about the protein being analyzed (you get more detail

about the amino acid sequence).

➢ Note: the peptides’ speed of travel is dependent on two variables:

✓ Charge (the peptides get ionized and thus might be singly-charged, doubly-

charged, triply-charged…etc).

✓ Mass.

(Each peptide travels according to its mass-to-charge ratio)

➢ Note: protein samples often contain mixtures of many different proteins which

makes it harder to identify proteins. To enhance the results of mass

spectrometry, we digest all of the proteins present in the sample (by trypsin) and

use chromatography techniques to separate the resulting peptides. After that,

we slowly inject them into the mass spectrometer. Some of the peptides are then

selected to proceed to the second mass spectrometer (tandem mass

spectrometry).

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❖ Protein Arrays:

✓ Protein biochemists developed an instrument with a similar concept to DNA

microarrays but specifically designed for proteins, called “protein arrays”.

✓ However, protein microarrays are more

complex than DNA microarrays because:

• Proteins are more complex than DNA

(DNA is composed of only 4 nucleotides

while proteins have more diverse

structures as they consist of 20 amino

acids and undergo various

modifications like glycosylation)

• DNA does not change over time (your

DNA in the morning is the same as your

DNA at night), while protein expression

differs with time depending on individual and environmental

circumstances, and this affects their detection (high variability).

➢ Types of Protein Arrays:

A. Expression Arrays: these microarrays reveal whether a protein is present in

a sample or not, and they include:

1. Forward-phase microarray

➢ In this array, we have a solid surface

containing many spots, and each spot

contains a different type of antibody that is

specific to a certain protein (the same spot

contains multiple copies of the same

antibody). Then, we add our sample (which

contains a mixture of proteins) to the

microarray and we detect the proteins. The spots which emit a signal indicate

that our sample contains the protein that corresponds to that spot.

➢ Note: The antibodies here are analogous to the probes in the DNA microarray.

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2. Reverse-phase microarray

➢ The concept of this microarray is somehow

the reverse of the previous type. Here, each

spot contains a different sample (samples

are probably taken from different patients

or different tissues..etc). Then we add one

type of labelled antibodies (specific to one

protein) to the entire microarray. The spot

that gives a signal indicates that the sample

in that spot contains the specific protein targeted by the antibodies added.

➢ EXTRA: A comparison between forward-phase and reverse-phase microarrays to

simplify them

Forward-phase microarray

We are detecting the expression of MULTIPLE proteins in ONE sample

Each spot contains a different type of antibody.

We add our one sample to the entire microarray and then start detection.

Reverse-phase microarray

We are detecting the expression of ONE protein in MULTIPE samples

Each spot contains a different sample. (each sample contains a mixture of proteins)

We add one type of antibodies to the entire microarray and then start detection.

B. Functional microarrays: these microarrays serve to reveal the types of

protein-protein, protein-DNA, protein-RNA, … etc interactions that a protein

makes, or the enzymatic activity of a certain protein. They include:

1. Interaction microarray:

➢ Each spot in this microarray contains a

different and known DNA sequence. Then

we add our protein of interest to the

microarray, and we can determine which

DNA sequences this protein interacts with.

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➢ Instead of DNA sequences, we can have different proteins in each spot, and then

we add our protein of interest to see with which proteins it interacts.

2. Enzymatic Microarray

➢ Each spot in this microarray contains a

different substrate, and then you add one

type of enzyme to determine which

substrate it acts on out of all the substrates

spotted in the microarray.

Recombinant DNA-based Molecular Techniques

➢ Recombinant DNA: a piece of DNA formed by joining DNA molecules from

different sources using molecular techniques. Example of recombinant DNA: a

plasmid (bacterial DNA) in which we inserted the insulin gene (human DNA).

❖ Cloning Vectors:

➢ Vector: a DNA molecule that can

act as a carrier of foreign genetic

material into another cell such as

plasmids (plasmids are circular

bacterial DNA molecules)

➢ Cloning Vectors are vectors that

are inserted into cells in order to

clone/amplify the number of

copies of a certain piece of DNA.

An example is: Cloning plasmid

vectors.

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➢ Cloning vectors contain at least three essential parts for DNA cloning:

1. Origin of replication (they can replicate independently).

2. Selectable marker-antibiotic resistance gene (it assists in isolation/separation

of bacterial cells that contain the cloning vector from bacterial cells that do

not)

✓ EXTRA: How does this separation occur?

We add the modified plasmids which contain the antibiotic-resistant

gene to the bacterial cells. Not all bacterial cells will take up this new

plasmid. In order to separate the cells that have this new plasmid from

cells that do not, we add the antibiotic to all of the cells. Only bacterial

cells that possess the recombinant plasmid would survive since they

have the antibiotic-resistant gene. All other cells die, leaving us with the

cells that have the plasmid.

3. Insertion sites (parts that allow the insertion of a foreign DNA fragment)

(EXTRA: these parts are often restriction sites that allow restriction enzymes

to cut and then we insert our DNA of interest).

❖ Expression Vectors:

• These vectors follow the same concept of cloning vectors, but the difference is

that:

✓ Cloning vectors are used to insert DNA fragments of interest into host cells so

that they make many copies of this DNA fragment. (so that whenever we want to

study this DNA fragment later on, we would have sufficient supply of it).

✓ Expression vectors are used to insert DNA fragments of interest into host cells so

that they express this DNA fragment and produce gene products (proteins).

Therefore, we find that expression vectors contain additional DNA parts

associated with transcription and translation like promoters and the Shine-

Dalgarno sequence, as they allow the host cell to express the gene and not

merely replicate it.

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• Expression vectors contain:

1. Promoter sequences upstream of the

gene to be inserted.

✓ The promoter does not have to be the

same promoter for the gene to be

inserted. It can be any promoter (We

can trick cells into expressing whatever

gene we want, as long as the promoter

is inserted upstream of the gene. Cells

do not know what gene is being

expressed, they mainly care about the

presence of the promoter).

2. Ribosomal binding sequences (Shine-

Dalgarno [SO] sequences)

3. An insertion (cloning) site.

4. A selectable marker (antibiotic resistance gene)

5. An origin of replication.

• Note: notice that the expression vectors contain the same parts as the cloning

vectors, but have additional parts like promoters and the SO sequence.

Remember that while we want the DNA of interest to be expressed when using

expression vectors, we still also want it to be replicated to further increase the

copies and thus even more expression could take place.

• After our gene of interest is expressed, we can purify the proteins and use them

to treat patients.

• Examples of proteins produced using this method: insulin, growth hormone,

plasminogen activator (for blood clotting), erythropoietin (used for maturation of

RBCs).

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• There are limitations to using bacterial cells as host cells for our expression

vectors:

1. Bacteria cannot form disulfide bonds (internal disulfide bonds in bacteria).

2. There is no post-translational modification of proteins like glycosylation.

3. Misfolding (bacterial cells do not have chaperones to fold proteins

resulting in misfolded proteins).

4. Degradation of proteins due to misfolding (previous point).

• Solution: use a eukaryotic system such as yeast cells.

➢ Yeast cells are:

✓ Single-cell organisms

✓ Simple eukaryotes

✓ They can behave like bacteria (they grow fast)

✓ They have eukaryotic systems similar to those of human cells (they can

do the things that our cells can do).

❖ Promoter analysis: Role of enzymes

• There are genes that are expressed differently in different conditions. If we want

to know how the promoter of a particular gene is regulated, or what sequences

are important in regulating a certain gene, we can use different systems. An

important example is the use of “fireflies” (which are insects that emit light) to

study the activity of a gene at certain conditions or elucidate the function of

certain regions of the promoter.

➢ Where does this light come from?

✓ A reaction takes place within the

bodies of fireflies, which involves

the conversion of D-Luciferin into

Oxyluciferin with the help of the

enzyme Luciferase. Energy is

released during the process in the

form of light.

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• Scientists took advantage of this phenomenon in fireflies: The intensity of the

light emitted from a firefly can be used as an indicator of the amount of

expression of the Luciferase gene (the more the expression, the higher the

intensity of light). So, by introducing different promoters of interest upstream of

the Luciferase gene, and cutting different sequences from the promoter, we can

know which sequences affect expression and which do not by monitoring the

intensity of light emitted after each experiment.

• Scientists designed an expression vector, where we insert the luciferase gene into

the vector, and upstream of this gene we insert the promoter we wish to study.

✓ If this promoter is inactive, no luciferase is

expressed, and thus no light would be

detected.

✓ If the promoter is slightly active, a little

amount of luciferase is produced, and weak

light could be detected.

✓ If the promoter is very active, there will be

a lot of luciferase and a lot of light would

be detected.

• Note: A reporter gene: a gene that helps in reporting results about something.

The luciferase gene is a “reporter gene”, because it “reports” the results of our

experiment in a visible manner (light intensity) (In other words, it indicates how

much expression occurred, this “report” is in the form of light intensity).

• In this experiment, we are not studying the luciferase gene, we are studying the

promoter’s activity in different circumstances.

• Extra note: D-Luciferin is added to the cells we are studying to guarantee that

there is enough substrate for the reaction to occur and produce light.

Summary: The promoter of the gene only is placed upstream of a “reporter gene” such as the luciferase gene in a plasmid, the plasmid is transfected (inserted) into the cells, and the expression level of luciferase (instead of the gene itself) is measured.

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➢ The promoter has

different regions:

✓ The core promoter.

✓ The activating region.

✓ The repressor region.

➢ Different experiments

✓ No promoter: there

should be almost no

expression (we notice

a very minimal

amount of expression which is normal due to “leakage”). (negative control)

✓ A good promoter: we add a promoter that we know is active and functional, thus

we notice that there is a good amount of expression. (positive control: meaning

that we use this promoter -which we know is active and functional and will give

positive results- just to make sure our experiment is being implemented correctly

and that the genetic system is fine so that other experiments can take place)

✓ The complete promoter: we add the entire promoter of interest containing all its

original parts (core, activating and repressor regions). We notice that we have

expression of the gene.

➢ In the following experiments, we start to manipulate the DNA by chopping off

parts of our promoter of interest:

✓ We start removing parts of the promoter in an orderly fashion: at first, we

notice that the more we remove from a certain part of the promoter, the greater

the increase in the amount of expression. We deduce that the region from which

we removed DNA must be a repressor region.

✓ We remove even more of the promoter: we notice a drop in the amount of

expression. We conclude that we have now started to remove DNA from the

activating region.

✓ We further remove parts of the promoter (core promoter) and we notice that

there is even less expression (hardly any expression).

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❖ Protein tagging or creation of protein hybrids:

➢ A tag is basically a label which allows us to detect

or identify something.

➢ Tags can be introduced into proteins to facilitate

their detection.

➢ How do we tag proteins?

✓ A protein-encoding gene (of interest) is inserted

into a special vector containing a tag gene, so

when the gene is expressed, the tag is expressed

along with it. The result is the production of a

protein with an extra sequence of amino acids

called tags.

✓ These tags allow easy protein purification and

detection.

➢ How do we benefit from these tags?

• As mentioned before, it helps in detection as in immunoblotting for instance.

• Revision of immunoblotting: SDS-PAGE Transfer to a membrane add

antibodies detection.

• BUT here, our antibodies do not target the protein itself, but instead target

the tag that is attached to it.

• Additionally, we can use these tags to purify proteins using affinity

chromatography.

(the antibodies that

target the tag are

attached to the

beads, and proteins

that contain the tag

would bind to the

antibodies, while

other proteins are

washed away).

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➢ Comments on the previous table:

✓ The ones in red are the ones we will focus on (GFP, GST, Poly-His)

✓ There are different tags, some are small like the Poly-His tag (which

consists of 6 Histidines. So, the tagged protein would start with 6

Histidines, and this tag does not affect the rest of the structure or function

of the protein)

✓ Some tags may be large like GFP and GST.

➢ His tag:

✓ We can use affinity chromatography

to purify proteins that have His tags.

(the addition of six histidines to a

protein would allow for purification

using beads with bound nickel

ions).

✓ Notice the image of gel

electrophoresis to the right.

Different types of proteins appear in

samples 1 and 2. While in sample 3,

only one protein band is shown due

to purification of the protein of

interest with His tags.

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❖ Purification of GST tagged proteins:

➢ Protein hybrid: a protein that consists

of multiple different proteins.

➢ Example: GST-tagged proteins.

➢ Glutathione S Transferase (GST) is an

enzyme that binds to a specific

substrate which is glutathione (a

tripeptide).

➢ We can design beads with attached

glutathione residues, and when our

sample passes through the beads, only

GST-tagged proteins will attach to the

beads. Other proteins are washed

away.

➢ Then, we can release the GST-tagged

proteins by adding free glutathione so the that the GST-tagged proteins bind to it

and move along with it and are then collected.

❖ GFP-tagged proteins:

➢ Another example of protein hybrids is: GFP-tagged proteins. ➢ GFP: Green Fluorescent Protein. It is a protein that comes from jellyfish (these

living creatures give a fluorescent color due to production of GFP) and scientists

took advantage of this protein in tagging. They were also able to separate

proteins with other colors later on. ➢ The GFP portion of

the GFP-tagged

protein folds

independently of

the protein of

interest, but is

attached to it. (The

same concept as

domains). Both

proteins maintain

their function.

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➢ Remember: Domains are regions that contain supersecondary structures

(multiple secondary structures). They fold independently from the rest of the

protein and retain their function.

➢ GFP allows for protein detection rather than for purification purposes. (GFP is a

fluorescent protein which gives a green color, so GFP-tagged proteins would

obtain a green color which helps in labelling and detection).

➢ Examples of proteins that can be labelled with GFP: actin, tubulin, mitochondrial

proteins.

➢ Whole cells can also be labelled (like neurons, which allows us to see how they

are connected to each other and study neural networks).

➢ Whole animals can be labelled as well (their health is not harmed by the process).

➢ So there is a world of possibilities.

Make sure you continue to part 2 of this

sheet.

Good Luck!